The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Tobacco SABP2 has been shown to display high affinity for salicylic acid (SA) and methylsalicylate (MeSA) and plays an important role in SAR signal development. Using biochemical approach, SABP2 has been shown to demonstrate strong esterase activity in converting MeSA to SA. Recent study shows that tetra fluoroacetophenone, a synthetic analog of SA, competitively inhibits SABP2 esterase activity as well as suppresses SAR signal development in tobacco mosaic virus (TMV)-infected tobacco plants. Not much has been studied on the effect of pesticides on plant defenses. Because both AChE and SABP2 are esterase-like proteins belonging to α/β hydroxylase superfamily, we hypothesize that pesticides may inhibit the MeSA esterase activity of SABP2 and block SAR development. Biochemical and molecular biology techniques were used to test this hypothesis. SAR in tobacco-TMV plant-pathogen system is measured by significant decrease in TMV-induced lesion sizes in secondarily inoculated distal leaves.

The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides or “conotoxins”. Conotoxins are bioactive peptides found in the venom duct of Conus spp. Like other neuropeptides, conotoxins are expressed as propeptides that undergo posttranslational proteolytic processing. Peptides derived from propeptides are typically cleaved at a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys or Arg-Lys) by proteases found in secretory vesicles. However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes. As many of the substrate-specific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible. In order to understand more about the substrate specificity of conotoxin substrate-specific proteases a characterisation study of one such endoprotease isolated from the venom duct of Conus textile was undertaken. The C. textile mollusc was chosen as a good source from which to isolate the endoprotease for two reasons; firstly, these cone shells are found in great abundance on the Great Barrier Reef (Queensland, Australia) and are readily obtainable and secondly, a number of conotoxin precursors and their cleavage products have been previously identified in the venom duct. In order to purify the endoprotease an activity-guided fractionation protocol that included a para-nitroanilide (p-NA) substrate assay was developed. The p-NA substrate mimicked the cleavage site of the conotoxin TxVIA, a member of the C. textile O-superfamily of toxins. The protocol included a number of chromatographic techniques including ion exchange, size-exclusion and reverse-phased HPLC and resulted in isolation of an active protease, termed Tex31, to >95% purity. The purification of microgram quantities of Tex31 made it possible to characterise the proteolytic nature of Tex31 and to further characterise the O-superfamily conopeptide propeptide cleavage site specificity. Specificity experiments showed Tex31 requires a minimum of four residues including a leucine in the P4 position (LNKR↓) for efficient substrate processing. The complete sequence of Tex31 was determined from cDNA. A BLAST search revealed Tex31 to have high amino acid sequence similarity to the CAP (abbreviated from CRISP (Cysteine-rich secretory protein), Antigen 5 and PR-1 (pathogenesis-related protein)) superfamily and most closely related to the CRISP family of mammalian and venom proteins that, like Tex31, have a cysteine-rich C-terminal domain. The CAP superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in processes as diverse as human brain tumour growth and plant pathogenesis. This is the first report of a biological role for the N-terminal domain of CAP proteins. A homology model of Tex31 constructed from two PR-1 proteins, Antigen 5 and P14a, revealed the highly conserved and likely catalytic residues, His78, Ser99 and Glu115. These three amino acids fall within a structurally conserved N-terminal domain found in all CAP proteins. It is possible that other CAP proteins are also substrate-specific proteases. With no homology to any known proteases, Tex31 may belong to a new class of protease. The sequence alignment of five Tex31-like proteins cloned from C. marmoreus, C. litteratus, C. arentus, C. planboris, and C. omaria show very high sequence similarity to Tex31 (~80%), but only one weakly conserved serine residue was identified when the conserved residues of the new Tex31-like protein sequences were aligned with members of the CAP superfamily. Future work to identify members of catalytic diad or triad, e.g. by site-directed mutagenesis, will rely on the expression of active recombinant Tex31. In this study neither Escherichia coli nor Pichia pastoris expression systems yielded active recombinant Tex31 protein, possibly due to the number of cysteine residues hindering the expression of correctly folded active Tex31. This study has shown Tex31 to be highly sequence specific in its cleavage site and it is likely that this high substrate specificity has confounded previous attempts to identify the proteolytic nature of other CAP proteins. With the proteolytic nature of one member of the CAP protein family confirmed, it is hoped this important discovery may lead the way to discovering the role of other CAP family members.